CN104620993B - A kind of method of degeneration African Chrysanthemum rejuvenating - Google Patents

Abstract

The present invention relates to a method for rejuvenating and rejuvenating decayed plants, in particular to a method for rejuvenating and rejuvenating degenerative gerbera. Roots of degenerated gerbera are used as explants, and after cultivation, the roots near the base of the root system are selected to dedifferentiate and form calluses. The part that organizes and forms adventitious buds is subjected to adventitious bud induction, multiplication culture, and rooted seedlings are obtained. The renewal and rejuvenation method of the present invention has strong growth potential of plants at the seedling stage, eliminates the effect of land continuous cropping on the growth and quality of gerbera seedlings, improves land use efficiency, enables reutilization of gerbera germplasm materials with degraded traits, and saves African chrysanthemum germplasm materials. The cost of chrysanthemum seedling production companies in purchasing high-quality seedlings.

Description

A method for regeneration and rejuvenation of degenerative gerbera

technical field

The invention relates to a method for rejuvenating and rejuvenating decayed plants, in particular to a method for rejuvenating and rejuvenating degenerative gerbera.

Background technique

Gerbera jamesonii Bolus ( Gerbera jamesonii Bolus), also known as gerbera, sunflower, etc., is a perennial hairy herbaceous perennial plant of the genus Gerbera in the family Asteraceae, native to Africa. Because of its huge flowers, tall and straight pedicels, rich flower colors, high rate of cut flowers, long life of flower arrangement, high ornamental value, and annual supply of fresh cut flowers under suitable conditions, it has become one of the top five cut flowers in the world. The genetic segregation phenomenon of gerbera in seed propagation is obvious, so that its excellent traits cannot be stably maintained between generations, and the multiplication coefficient of ramet propagation is low, which cannot meet the market's demand for seedlings. The current commercially produced gerbera varieties all use Tissue culture technology for rapid propagation of seedlings.

With the improvement of people's living standards, my country's demand for fresh cut flowers of gerbera and seedlings of fine varieties of gerbera is increasing day by day. According to statistics, the national gerbera planting scale increased from 32,700 mu in 2006 to 81,000 mu in 2012, with an average annual growth rate of 21%. However, the selection and breeding of excellent varieties of gerbera in my country is relatively lagging behind, and the number of excellent varieties with independent intellectual property rights is almost zero. The excellent varieties of gerbera cultivated in bulk commercialization are completely dependent on foreign purchases, and then use tissue culture technology to propagate seedlings. supply the domestic market. Correspondingly, there are many research reports on tissue culture methods of gerbera in China, mainly focusing on the type of medium, selection of explants, types of hormones and their ratios, induction of rooting, and transplanting and hardening of seedlings. In order to expand the scope of gerbera tissue culture explants, many researchers have screened and studied the explant materials of different organs of gerbera. Calyx, receptacle, filament, leaf, stem segment, petal, ovary, pedicel, Flower buds, stem tips, etc. established a tissue culture technology system for explants, and obtained regenerated plants. Through the investigation of gerbera seedling production companies and flower growers, it was found that the tissue culture seedlings using the above-mentioned gerbera plant organs as explants, after 2 to 3 years of subculture, the seedlings degenerated, mainly It is characterized by slow growth at the seedling stage, short and thin pedicels, smaller flower shape, lighter flower color, and loss of commodity value; second, continuous cropping obstacles after seedling planting are common, affecting yield and quality. Therefore, producers of gerbera seedlings have to purchase seedlings of the same variety from abroad again every 2 to 3 years for production, which is one of the main reasons why the current cost of gerbera seedlings remains high.

The methods of plant renewal and rejuvenation mainly include sexual reproduction and rejuvenation, root sprouting or stem-based sprouting, repeated pruning, young stock grafting, continuous cutting and tissue culture. Among them, the sexual reproduction and rejuvenation method is to realize the rejuvenation of high-quality materials through seed propagation; the root sprouting strip or stem-based sprouting strip method is to utilize the adventitious buds or dormant buds in the juvenile stage in the roots and trunk bases, and promote germination by burying roots or surrounding them. Cutting, engraving, etc. to promote germination, and further cutting propagation after obtaining sprouts, so as to achieve the purpose of rejuvenation; repeated pruning method is to maintain the physiological state of the trunk through intensive pruning; young stock grafting method is to graft the old scion On the young rootstock, use the juvenile physiological state of the rootstock to promote the rejuvenation of the scion; the continuous cutting method refers to cutting the branches taken from the old trees, and then cutting the surviving plants, so reciprocating, after Repeated cuttings for several years can significantly improve the rooting condition of cuttings; the regenerated plants obtained by tissue culture can achieve the purpose of rejuvenation and rejuvenation due to dedifferentiation. Among the above-mentioned plant renewal and rejuvenation methods, the root sprouting method or stem-based sprouting method, repeated pruning method, young stock grafting method, and continuous cutting method are mainly applicable to perennial woody plants, while sexual reproduction and rejuvenation and tissue culture methods are used in woody and It can be applied to herbaceous plants, but the seed propagation is prone to separation, so that the excellent traits cannot be maintained stably. The explant materials used in tissue culture are different, and there are also position effects, maturation effects, etc., and multi-generational subculture in tissue culture Post-cultivation degradation is common.

Contents of the invention

In order to solve the problem of trait degeneration of seedlings obtained by mainly using aboveground parts such as flower organs, leaves and stem tips as gerbera explants in the prior art, the present invention proposes a method for rejuvenating and rejuvenating degenerated gerbera.

A kind of degenerative gerbera regeneration and rejuvenation method of the present invention is characterized in that it is implemented according to the following steps:

(1) Obtaining the roots of degenerated gerbera: using degenerated gerbera aseptic seedlings as raw materials, cut the aseptic seedlings with a height of about 3 to 4 cm into individual seedlings, and inoculated them in the culture medium. The formula is 1/2MS + IBA 0.2 ~ 1.0 mg/L, pH 5.8 ~ 6.2, sucrose 20 ~ 30 g/L, agar 4 ~ 7 g/L; the culture temperature is 21±2℃, and the light intensity is 1600~1800 The light of Lux was 12 h/d; when the root grew to about 3 cm, the root segments of 0.5-1.0 cm were cut from the base of the root system to the tip of the root system in sequence according to the base of the root system, the middle of the root system and the root tip as explant materials;

(2) Screening of root segments: After rinsing the intercepted root base, middle root and root tip with a length of 0.5 ~ 1.0 cm under sterile distilled water, they were inoculated on the starting medium for cultivation. MS + 6-BA0.5 ~ 1.5 mg/L+NAA 0.1 ~ 1.0 mg/L, pH 5.8 ~ 6.2, sucrose 20 ~ 30 g/L, agar 4 ~ 7 g/L; culture temperature 21±2℃, Cultivate for 25 to 30 days with a light intensity of 1600 to 1800 Lux and 12 h/d;

(3) Induction of adventitious buds: intercept the root base of degenerated gerbera aseptic tissue culture seedlings with a length of about 0.5 ~ 1.0 cm, wash them under distilled water, inoculate them on the adventitious bud induction medium, and culture them. The formulation of the medium is MS + 6-BA 0.5 ~1.5 mg/L+NAA 0.1 ~ 1.0 mg/L, pH 5.8 ~ 6.2, sucrose 20 ~ 30 g/L, agar 4 ~ 7 g/L; culture conditions are temperature 21±2℃, light The intensity was 1600 ~ 1800 Lux, the light time was 12 h/d, and the culture was 25 ~ 30 days; observe the formation of callus and adventitious buds in different parts of the root segment, and select the root segment near the base of the root system to dedifferentiate and form callus, and form parts of adventitious buds;

(4) Proliferation culture: transfer the 1-2 cm adventitious buds with good growth induced by the basal root segment to the proliferation medium. The formula of the proliferation medium is: MS + 6-BA 0.5-2.0 mg/L + NAA 0.05 ~ 0.2 mg/L, pH 5.8 ~ 6.2, sucrose 20 ~ 30 g/L, agar 4 ~ 7 g/L; culture temperature is 21±2°C, light intensity is 1600 ~ 1800Lux 12 h/d , cultivated for 25 to 30 days to form clustered shoots with a height of 2 to 3 cm; the formed clustered shoots can be cut into single buds, and placed on the proliferation medium for proliferation culture;

(5) Rooting culture: when the clustered bud plants obtained from multiplication grow to 2 ~ 3 cm, cut the clustered buds into 2 ~ 4 leaves to form a single bud, and inoculate them with a formula of 1/2MS + IBA 0.2 ~ 1.0 mg/L, add 20 ~ 30 g/L sucrose, and culture on rooting medium of 4 ~ 7 g/L agar, the culture temperature is 21±2℃, and the light intensity is 1600 ~ 1800Lux for 12 h/d; After culturing for 25-30 days, rooted gerbera seedlings with a root length of 1.5-2.5 cm were cultivated;

(6) Seedling hardening and transplanting: When the roots of the rooted seedlings are more than 2 cm long, harden the seedlings indoors at a room temperature of 25°C for 2 to 4 days, take out the rooted seedlings, wash the medium adhered to the roots, and put The seedlings are sent to the general gerbera seedling cultivation bed for transplanting.

The observation results of root segment screening are shown in Table 1.

.

The development degree of perennial plants increases sequentially from the base to the top, and there are maturity effects, position effects, and age effects in some of the aboveground organs, which can easily cause aging of asexually propagated seedlings such as cuttings, layering, grafting, and tissue culture, thereby affecting the growth of seedlings. In the vegetative propagation of perennial tree species, in order to maintain the juvenile nature of seedlings and promote the growth of seedlings, generally by cutting the base of the trunk to promote germination, collecting sprouts as propagation materials for seedling cultivation, especially the root system of plants, there is no stage development Sex is always juvenile, so there is no staged aging. Therefore, the seedlings obtained by propagating with roots as the starting material can obtain the same juvenile nature as the seedlings, so as to realize the rejuvenation and rejuvenation effect of the decayed tree species and restore their growth vigor. Gerbera belongs to herbaceous plants, and the root system is small, so it is impossible to rejuvenate and rejuvenate by burying the roots to promote germination. Only the root section is selected as explants, and the method of tissue culture is used for rejuvenation and rejuvenation. Drawing on the method of rejuvenation and regeneration of perennial arbor species, the tissue culture regeneration system of degenerated gerbera with root segments as explants was established to restore its degraded traits and commercial value, which can not only greatly save the cost of purchasing raw materials for propagation, but also provide long-term effective Safer preservation of gerbera reproductive material provides a new way.

The seedlings that had been cultivated for 2 months were transferred to the flower farm base engaged in the production of fresh-cut gerbera flowers. New land and 3-year continuous cropping land were selected for planting. The row spacing between plants and rows was 30 cm × 50 cm, and the daily management was carried out according to the general management method. According to observations and statistics, the survival rate of seedlings planted on new land reached over 98%, and the survival rate of seedlings planted on 3-year continuous cropping land reached over 95%. Flowering, pedicel length, thickness and straightness, corolla size, etc. are all restored, and its commercial value has been restored.

See Table 2 for the recovery effect of degenerated traits of Gerbera chrysanthemum tissue culture seedlings with root segments as explants.

Note: The leaf length and leaf width of the seedlings are taken as the representative values of seedling growth, the former value is the leaf length, and the latter value is the leaf width.

The method for rejuvenating and rejuvenating degenerative gerbera of the present invention has the following advantages:

Compared with the tissue-cultured seedlings of the same species imported from abroad recently with filaments as explants, the gerbera tissue-cultured seedlings with basal root segments as explants grew vigorously at the seedling stage and were released for sale 30 days earlier. The gerbera seedling production company saves production costs and improves the utilization efficiency of nursery land.

Gerbera chrysanthemum seedlings produced by explant tissue culture with base root section can overcome the obstacles of land continuous cropping, eliminate the effect of land continuous cropping on the growth and quality of gerbera chrysanthemum seedlings, save the cost of constantly replacing land for flower farmers, and also improve land use efficiency.

Gerbera chrysanthemum seedlings with degraded traits were used as raw materials, and the base root section was used as explant material for tissue culture and rapid propagation. After flowering, the gerbera chrysanthemum seedlings obtained had elongated and thickened pedicels and larger corolla, which were similar to those obtained from The traits are consistent when imported from abroad, the commodity value of the degenerated gerbera can be restored, the renewal and rejuvenation of the degenerated gerbera can be realized, the germplasm materials of the degraded gerbera can be reused, and the gerbera seedling production company can save the cost of purchasing high-quality seedlings. aspects of the cost.

detailed description

Embodiment 1: Below in conjunction with example the technical scheme of the present invention is further described. The implementation time is from 2013 to 2014. The adventitious bud induction, plant regeneration and seedling hardening of gerbera were carried out in the tissue culture laboratory of the School of Forestry, Southwest Forestry University. The tissue culture seedlings were transplanted and seedlings were cultivated in Meilan flower seedlings It is carried out by a limited liability company, and the seedling planting and fresh-cut flower production are carried out at the gerbera planting base in Jinning County.

(1) Obtaining the roots of degenerated gerbera: the aseptic bottle seedlings (variety name solar wind) propagated with gerbera filaments as explants by Kunming Meilan Flower Seedling Co., Ltd. as the original material, the material is After 3 years of subculture and breeding, the seedlings have shown degenerate traits, that is, the seedlings grow slowly, the pedicel becomes shorter, and the flower type becomes smaller, which cannot meet the requirements of gerbera commercial seedlings and are sold. On the ultra-clean workbench, cut the aseptic seedlings with a height of about 3 to 4 cm into individual seedlings, and inoculate them on the medium of 1/2MS+IBA 0.5 mg/L for rooting and sampling. Among them, pH 5.8, sucrose 20 g/L, agar 4 g/L. Culture conditions: The culture temperature is 21±2°C, and the light intensity is 1600-1800 Lux for 12 h/d. When the root grows to about 3 cm, 0.8 cm root segments are cut from the root base to the root tip according to the root base, root middle and root tip in turn as explant materials;

(2) Screening of root segments: The base, middle and tip of the root system with a length of 0.8 cm were intercepted on the ultra-clean workbench, rinsed with sterile distilled water, and then inoculated on the starting medium for cultivation. The medium formula is MS + 6-BA 0.5 ~ 1.5 mg/L + NAA 0.1 ~ 1.0 mg/L, pH 5.8 ~ 6.2, sucrose 20 ~ 30 g/L, agar 4 ~ 7g/L; culture temperature is 21±2 ℃, with a light intensity of 1600-1800 Lux for 12 h/d, culture for 25-30 days, and observe the formation of calluses and adventitious buds in different parts of the roots. The results showed that only the root segment near the base of the root system could dedifferentiate to form callus and form adventitious buds, while the middle and root tip parts could not produce callus. Therefore, the root base was selected as explants for tissue culture of degenerative gerbera;

(3) Induction of adventitious buds: Cut off the root base of degenerated gerbera aseptic tissue-cultured seedlings with a length of 0.8 cm, rinse them under distilled water, inoculate them on adventitious bud induction medium, and culture them. The formulation of the medium is MS + 6-BA 0.5 ~1.5 mg/L+NAA 0.1 ~ 1.0 mg/L, pH 5.8 ~ 6.2, sucrose 20 ~ 30 g/L, agar 4 ~ 7 g/L; culture conditions are temperature 21±2℃, light The intensity is 1600 ~ 1800 Lux, the light time is 12 h/d, and the culture is 25 days;

(4) Proliferation culture: transfer the 1-2 cm adventitious buds with good growth induced by the basal root segment to the proliferation medium. The formula of the proliferation medium is: MS + 6-BA 0.5-2.0 mg/L + NAA 0.05 ~ 0.2 mg/L, pH 5.8 ~ 6.2, sucrose 20 ~ 30 g/L, agar 4 ~ 7 g/L; culture temperature is 21±2°C, light intensity is 1600 ~ 1800Lux 12 h/d , cultivated for 25 days, and obtained clustered buds with a height of 2 ~ 3cm. Repeatedly cutting the clustered buds formed into single buds and inoculating them on the proliferation medium for proliferation culture can make Gerbera chrysanthemum proliferate rapidly and in large quantities, and its value-added multiple reaches 5.07 times;

(5) Rooting culture: when the clustered bud plants obtained from multiplication grow to 2 ~ 3 cm, cut the clustered buds into 2 ~ 4 leaves to form a single bud, and inoculate them with a formula of 1/2MS + IBA 0.2 ~ 1.0 mg/L, add 20 ~ 30 g/L sucrose, and culture on rooting medium of 4 ~ 7 g/L agar, the culture temperature is 21±2℃, and the light intensity is 1600 ~ 1800Lux for 12 h/d; After 25 days of cultivation, rooted gerbera seedlings with a root length of 1.5 to 2.5 cm were obtained, and the rooting rate reached 100%;

(6) Seedling hardening and transplanting: When the roots of the rooted seedlings are more than 2 cm long, open the bottle cap and harden the seedlings in a room with a room temperature of 25°C for 2 to 4 days, take out the rooted seedlings, and clean the roots that adhere to the roots. Send the seedlings to the seedling company for transplanting on the common gerbera seedling cultivation bed. After transplanting, the temperature in the greenhouse is between 25 and 30°C, the humidity is between 75 and 80%, and spraying for 3 days Water once, survive after 10-15 days. The transplanting survival rate of the seedlings reached 98.2%, and after two months of cultivation, the height of the seedlings reached more than 10 cm, which met the requirements for the sale of gerbera seedlings;

(7) Detection of the recovery effect of degraded traits: Transfer the seedlings with a seedling height of more than 10 cm after 2 months of cultivation to the flower farm base for fresh-cut gerbera production in Jinning County, and select a new field seedbed with a width of 120 cm and a 3-year continuous cropping seedbed to carry out the test. Planting, the spacing between plants and rows is 30 cm × 50 cm, and the daily management is carried out according to the general management method. According to observations and statistics, the survival rate of seedlings planted on new land reached over 98%, and the survival rate of seedlings planted on 3-year continuous cropping land reached over 95%. Flowering, the average length of the pedicel is 42.21 cm, the average thickness is 0.74 cm, straight, and the average corolla size is 9.45 cm, which meets the requirements of fresh cut gerbera flowers and restores its commercial value.

Claims (1)

1. a kind of method of degeneration African Chrysanthemum rejuvenating, it is characterised in that follow these steps to implement：

（1）The acquisition of degeneration African Chrysanthemum root segment：Using the sterile bottle seedling of rudimentary African Chrysanthemum as original material, by high 3 ~ 4 cm's Aseptic seedling is cut into individual plant seedling, is inoculated in culture medium, and culture medium prescription is 1/2MS+IBA 0.2 ~ 1.0 mg/L, pH 5.8 ~ 6.2, the g/L of sucrose 20 ~ 30, the g/L of agar 4 ~ 7；Cultivation temperature is 21 ± 2 DEG C, using intensity of illumination as 1600 ~ 1800 The Lux h/d of illumination 12；When root growth to 3 cm or so, root system base portion, root system are pressed successively from root system base portion to tip of a root part Middle part and the cm of tip of a root portion intercepts 0.5 ~ 1.0 root segment are used as explant material；

（2）The screening of root segment：It is 0.5 ~ 1.0 cm root system base portion, root system middle part and tip of a root part, nothing by the length of interception After being rinsed well under bacterium distilled water, be inoculated in primary culture medium and cultivated, culture medium prescription be MS+6-BA 0.5 ~ 1.5 mg/L+NAA 0.1 ~ 1.0 mg/L, pH 5.8 ~ 6.2, the g/L of sucrose 20 ~ 30, the g/L of agar 4 ~ 7；Culture temperature Spend for 21 ± 2 DEG C, the h/d of illumination 12 by 1600 ~ 1800 Lux of intensity of illumination, cultivate 25 ~ 30 d；Observe different parts Root segment produces callus and adventitious bud formation situation；As a result display only being capable of dedifferentiation formation callus close to the root segment of root system base portion Tissue, and adventitious bud is formed, and middle part and tip of a root part can not produce callus；Therefore selection root system base portion is used as explant Body enters the tissue cultures of row degradation African Chrysanthemum；

（3）The induction of adventitious bud：Under long 0.5 ~ 1.0 cm of the interception sterile tissue-cultured seedling root system base portion of degeneration African Chrysanthemum, distilled water After rinsing well, it is inoculated on adventitious bud induction culture base and is cultivated, culture medium prescription is MS+6-BA 0.5 ~ 1.5 Mg/L+NAA 0.1 ~ 1.0 mg/L, pH 5.8 ~ 6.2, the g/L of sucrose 20 ~ 30, the g/L of agar 4 ~ 7；Condition of culture is Temperature is 21 ± 2 DEG C, and the Lux of intensity of illumination 1600 ~ 1800, the h/d of light application time 12 cultivate 25 ~ 30 d；

（4）Multiplying culture：Obtained preferable 1 ~ 2 cm adventitious buds of growing way are induced to be transferred to proliferated culture medium base portion root segment On, the formula of proliferated culture medium is：0.5 ~ 2.0 mg/L+NAA of MS+6-BA 0.05 ~ 0.2 mg/L, pH 5.8 ~ 6.2, the g/L of sucrose 20 ~ 30, the g/L of agar 4 ~ 7；Condition of culture is 21 ± 2 DEG C of temperature, using intensity of illumination as 1600 ~ The 1800 Lux h/d of illumination 12, cultivates 25 ~ 30 d, forms the high Multiple Buds of 2 ~ 3cm；The Multiple Buds of formation are cut into list Bud, is placed on proliferated culture medium and carries out Multiplying culture；

（5）Culture of rootage：When breeding obtained Multiple Buds plant and growing to 2 ~ 3 cm, Multiple Buds are cut into 2 ~ 4 leaves Son is one plant of simple bud, and it is the mg/L of 1/2MS+IBA 0.2 ~ 1.0 to be inoculated into formula, additional 20 ~ 30 g/L sucrose, 4 ~ Cultivated on the root media of 7 g/L agar, cultivation temperature is 21 ± 2 DEG C, using intensity of illumination as 1600 ~ 1800 Lux light According to 12 h/d；Cultivate after 25 ~ 30 d, cultivate a length of 1.5 ~ 2.5 cm of root African Chrysanthemum rooted seedling；

（6）Hardening is with transplanting：When the root of offspring to be generated is up to 2 more than cm, it is 25 DEG C of indoor hardening 2 ~ 4 days in room temperature, takes Go out rooted seedling, clean the culture medium for sticking to root, seedling is delivered to and transplanted on general African Chrysanthemum seedling fostering seedbed；

（7）Degenerativ character recovery effects are detected：2 months heights of seedling will be cultivated to be planted in 10 more than cm seedling, seeding row spacing is The cm of 30 cm × 50, daily management is carried out by general way to manage.